Nitric Oxide Participates in the Stimulatory and Neurotoxic Action of Endothelin on Rat Striatal Dopaminergic Neurons

1. Our method of real-time monitoring of dopamine release from rat striatal slices revealed that endothelin (ET)-3-induced dopamine release was inhibited by N ^G-methyl-L-arginine (L-NMMA; 1 mM), an inhibitor of nitric oxide (NO) synthase, while N ^G-methyl-D-arginine (D-NMMA; 1 mM), an inactive isomer of L-NMMA, had no effect.2. The inhibition of L-NMMA (0.1 mM) became apparent when tissues were pretreated with tetrodotoxin (1 M) for 30 min and subsequently exposed to ET-3 (4 M).3. L-NMMA (0.1 and 1 mM) dose dependently protected against ET-3-triggered hypoxic/hypoglycemic impairment of striatal responses to high K⁺.4. Thus, NO may work as a promoter in mediation of the stimulatory and neurotoxic action of ET-3 on the striatal dopaminergic system, presumably by interacting with interneurons in the striatum.     

Activation of Protein Kinase C Suppresses the γ-Aminobutyric AcidB Receptor-Mediated Inhibition of the Vesicular Release of Noradrenaline and Acetylcholine

Modulation of the gamma-aminobutyric acidB (GABAB) receptor-mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation-evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the high K(+)-evoked Ca2+-dependent release of NA and ACh, but not the ouabain-evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic-vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K(+)-evoked release of NA and ACh, but not the ouabain-evoked Ca(2+)-independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor-mediated inhibition of high K(+)-evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation-evoked Ca(2+)-dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor-mediated response.     

Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding.

The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.     

Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents

The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.     

Age-Related Modulation of the Effects of Obesity on Gene Expression Profiles of Mouse Bone Marrow and Epididymal Adipocytes

This study aimed to characterize and compare the effects of obesity on gene expression profiles in two distinct adipose depots, epididymal and bone marrow, at two different ages in mice. Alterations in gene expression were analyzed in adipocytes isolated from diet-induced obese (DIO) C57BL/6J male mice at 6 and 14 months of age and from leptin deficient mice (ob/ob) at 6 months of age using microarrays. DIO affected gene expression in both depots at 6 and 14 months, but more genes were altered in epididymal than bone marrow adipocytes at each age and younger mice displayed more changes than older animals. In epididymal adipocytes a total of 2789 (9.6%) genes were differentially expressed at 6-months with DIO, whereas 952 (3.3%) were affected at 14-months. In bone marrow adipocytes, 347 (1.2%) genes were differentially expressed at 6-months with DIO, whereas only 189 (0.66%) were changed at 14-months. 133 genes were altered by DIO in both fat depots at 6-months, and 37 genes at 14-months. Only four genes were altered in both depots at both ages with DIO. Bone marrow adipocytes are less responsive to DIO than epididymal adipocytes and the response of both depots to DIO declines with age. This loss of responsiveness with age is likely due to age-associated changes in expression of genes related to adipogenesis, inflammation and mitochondrial function that are similar to and obscure the changes commonly associated with DIO. Patterns of gene expression were generally similar in epididymal adipocytes from ob/ob and DIO mice; however, several genes were differentially expressed in bone marrow adipocytes from ob/ob and DIO mice, perhaps reflecting the importance of leptin signaling for bone metabolism. In conclusion, obesity affects age-associated alterations in gene expression in both epididymal and bone marrow adipocytes regardless of diet or genetic background.     

Effects of colonization of a bacterial endophyte,Azospirillum sp. B510, on disease resistance in tomato

A plant growth-promoting bacteria, Azospirillum sp. B510, isolated from rice, can enhance growth and yield and induce disease resistance against various types of diseases in rice. Because little is known about the interaction between other plant species and this strain, we have investigated the effect of its colonization on disease resistance in tomato plants. Treatment with this strain by soil-drenching method established endophytic colonization in root tissues in tomato plant. The endophytic colonization with this strain-induced disease resistance in tomato plant against bacterial leaf spot caused by Pseudomonas syringae pv. tomato and gray mold caused by Botrytis cinerea. In Azospirillum-treated plants, neither the accumulation of SA nor the expression of defense-related genes was observed. These indicate that endophytic colonization with Azospirillum sp. B510 is able to activate the innate immune system also in tomato, which does not seem to be systemic acquired resistance.     

Phylogenetic Position of the Mitochondrion-Lacking Protozoan Trichomonas tenax,Based on Amino Acid Sequences of Elongation Factors 1α and 2

Major parts of amino-acid-coding regions of elongation factor (EF)-1α and EF-2 in Trichomonas tenax were amplified by PCR from total genomic DNA and the products were cloned into a plasmid vector, pGEM-T. The three clones from each of the products of the EF-1α and EF-2 were isolated and sequenced. The insert DNAs of the clones containing EF-1α coding regions were each 1,185 bp long with the same nucleotide sequence and contained 53.1% of G + C nucleotides. Those of the clones containing EF-2 coding regions had two different sequences; one was 2,283 bp long and the other was 2,286 bp long, and their G + C contents were 52.5 and 52.9%, respectively. The copy numbers of the EF-1α and EF-2 gene per chromosome were estimated as four and two, respectively. The deduced amino acid sequences obtained by the conceptual translation were 395 residues from EF-1α and 761 and 762 residues from the EF-2s. The sequences were aligned with the other eukaryotic and archaebacterial EF-1αs and EF-2s, respectively. The phylogenetic position of T. tenax was inferred by the maximum likelihood (ML) method using the EF-1α and EF-2 data sets. The EF-1α analysis suggested that three mitochondrion-lacking protozoa, Glugea plecoglossi, Giardia lamblia, and T. tenax, respectively, diverge in this order in the very early phase of eukaryotic evolution. The EF-2 analysis also supported the divergence of T. tenax to be immediately next to G. lamblia. Received: 15 February 1996 / Accepted: 28 June 1996